The interaction of major histocompatibility complex (MHC) antigens, and their cognate ligands, the T-cell receptor (TCR), plays a central role in the regulation of immune responses. Using systems developed during previous project periods, we will analyze the interactions of TCR and MHC molecules, antigen/MHC complex expression, and new therapeutic approaches to immunomodulation. While structural studies helped elucidate the interaction between TCR and MHC molecules, the basis of degenerate TCR recognition has not yet been answered. This question is important in understanding molecular mimicry, a model of autoimmune and alloreactive responses, and how low affinity interactions, such as peptide/MHC antagonists and positive selection, stimulate T-cells. We will use changes in: crosslinking patterns; extrinsic and intrinsic fluorescence; fluorescence energy transfer; and protease sensitivity to study structural aspects of the TCR/MHC interface and TCR signaling. The ability to identify and track antigen/MHC complexes has been hampered by the lack of high affinity specific reagents. Soluble divalent versions of TCR, which we have recently developed, have high affinity and specificity for peptide/MHC complexes. We will use these molecules to identify peptides involved in positive selection. We will also use divalent TCR to analyze the influence of lymphokines, GM-CSF, IFN-gamma, and TNF-alpha on antigen processing in vitro and expression in vivo during crosspriming. Soluble divalent versions of MHC and TCR molecules also have potential immunomodulatory application both in vitro and in vivo. Our earlier work showed that soluble divalent MHC inhibits alloreactive T-cell activation in vitro. During this last project period we have extended these finding and shown that soluble divalent MHC inhibits allograft rejection. Over the next several years we will further characterize the effects of soluble divalent MHC molecules on both alloreactive and antigen-specific immune responses. We will also see if soluble divalent TCR/Ig can modulate immune responses. We have produced divalent, high affinity soluble analogs of most of the components of the specific immune responses. To further increase the effective affinity, we will make soluble multivalent complexes of both TCR and MHC molecules. To more effectively recapitulate the TCR binding complex, we will incorporate the CD8 accessory molecule into soluble TCR analogs. An enhanced understanding of the interactions of MHC and TCR molecules will improve our understanding of their role in the generation of both normal and pathological immune responses. Soluble divalent MHC and TCR analogs also suggest potential novel immune-based therapeutic strategies.